Specific binding proteins (hereinafter referred to as "receptors") on the plasma membrane of a mammalian cell are essential for the interaction of the cell with the external environment. For example, receptors mediate the physiological actions of hormones, antibodies, enzymes, neorotransmitters and other biologically active substances. They are also responsible for the cellulor selectivity of drugs, toxins and poisons, and for the tissue tropism (growth response) of certain pathogenic viruses.
An antibody against a cell surface receptor (hereinafter referred to as an "anti-receptor antibody") can develop naturally in vivo, or can be induced by the deliberate immunization of a heterologous species with a purified receptor. In some cases, an anti-receptor antibody can stimulate the biological effects of a ligand or antigen recognized by the receptor. As used herein, a ligand is any substance that reacts with and binds to an antibody. An anti-receptor antibody inhibits, or reverses, the binding of a ligand to its complementary receptor without exhibiting intrinsic agonist activity.
The potential therapeutic applications of purified highly specific, anti-receptor antibodies are almost limitless. For example, the passive administration of antibodies against cellular receptors for pathogenic viruses can protect against infection by (a) binding to the viral receptor on susceptible cells and preventing virus penetration; and (b) simulating the molecular structure of the virus and inducing a specific anti-viral (anti-ligand) antibody response.
Moreover, in individuals exposed to microbial exotoxins and endotoxins, synthetic poisons, or toxic concentrations of commonly used drugs, anti-receptor antibodies can block the potential lethal pharmacologic action of the foreign agent (or antigen) by preventing its binding to cells. In some instances, a high affinity anti-receptor antibody may actually displace the toxin or drug from the receptor, thereby reversing any deleterious effects, and accelerating removal, of the toxin or drug from the body. Examples of instances in which such therapy can be life-saving include exposure to exotoxins or endotoxins, drug overdose, biological warfare, snake bite and the like.
In short, anti-receptor antibodies have a tremendous potential as therapeutic agents. However, before the present invention, such antibodies were extremely difficult and expensive to prepare. In particular, the generation of an anti-receptor antibody has heretofore required either the purification of the receptor to a condition near homogeneity, and inoculation of the essentially pure receptor into a heterologous species, and/or the screening of hybridoma antibodies derived from an animal host immunized with whole cells or semi-purified receptor preparations.
For many antigens including viruses, toxins, drugs and hormones, the specific receptors associated with the particular antigen have been well-characterized. The screening of hybridoma antibodies, however, is a time-consuming and expensive procedure. Moreover, in both instances described above, the specific anti-receptor antibody must be purified from sera or ascites (serous fluid that accumulates in the abdominal cavity). For these reasons, the development of anti-receptor antibodies for therapeutic applications has proceeded rather slowly relative to other areas of immunology.
By way of further background, an antibody is an immunoglobulin molecule that has a specific amino acid sequence by virtue of which it binds only with the antigen that induced its synthesis or with a closely related antigen. An immunoglobulin molecule includes two kinds of polypeptide chains -- a pair of larger identical polypeptide chains referred to as heavy chains and two identical smaller ones referred to as light chains. These polypeptide chains are held together by disulfide bonds and by noncovalent bonds, which are primarily hydrophobic. The heavy and light polypeptide chains are synthesized in vivo on separate ribosomes, assembled in the cell and secreted as an intact immunoglobulin molecule.
The understanding of the structure and function of immunoglobulins has been facilitated by studies of fragments produced by enzymatic cleavage of the antibody molecule. For example, treatment of an antibody molecule with the enzyme papain produces two antigen-binding fragments (designated "Fab") and a complement-binding fragment (designated "Fc"), which contains no antigen-binding capacity but determines important biological properties of the intact molecule.
Treatment of an antibody molecule with the enzyme pepsin, on the other hand, produces an antigen-binding fragment (F(ab)'.sub.2) and a somewhat smaller complement-binding fragment (Fc).
The amino-terminal half of the light (L) chains and the amino-terminal quarter of the heavy (H) chains of an immunoglobulin molecule vary in their amino acid sequence and are termed the variable regions (V regions) of the polypeptide chains. Portions of the V region of one heavy and one light polypeptide chain contribute the site for antigen or ligand binding. The constant region of H chains allows their differentiation into a class or subclass and confers to the immunoglobulins certain biological properties such as the ability to activate complement, cross the placenta and bind to polymorphonuclear leukocytes or macrophages.
Five immunoglobulin classes (IgG, IgA, IgM, IgD, IgE) are recognized on the basis of structural differences of their heavy chains including the amino acid sequence and length of the polypeptide chain. The antigenic determinants on the heavy chains also permit the identification and quantitation of the immunoglobulin classes by immunochemical assay techniques.
Such immunochemical assay techniques include radioimmunoassay (RIA) which establishes a competition between a known amount of a radioactive labeled antigen and an unknown amount of the same or a similar antigen present in a biological sample with a limited, standard amount of an antibody.
Another immunochemical assay technique is enyzme immumoassay (EIA) which can be even more sensitive than RIA methods. Four basic forms of EIA have been developed; the competitive binding, or enzyme-linked immunosorbent assay (ELISA) test, the immunoenzymometric test, the sandwich method for antigen or antibody, and the homogeneous EIA.
The ELISA test was the first to be developed and is patterned after the standard competitive RIA procedure. Labeled and unlabeled antigen compete for attachment to a limited quantity of solid-phase antibody. The enzyme label that is displaced is quantitated, and the calculations that follow are essentially the same as in RIA procedures.
In the immunoenzymometric procedure, an unknown quantity of antigen is reacted with an excess of enzyme-labeled antibody, and then solid-phase antigen is added. Centrifugation removes the antibody molecules that reacted with the solid-phase antigen, leaving enzymic activity in the soluble phase. The enzyme actively associated with the soluble phase is thereafter measured, and thereby provides a measure of the antigen concentration in the unknown sample.
The sandwich technique relies on the multivalence of antigen and its capacity to bind simultaneously with two molecules of antibody. The first antibody molecule is usually a solid-phase reactant. It is used in excess to ensure binding (complexation) of all the antigen molecules in the unknown sample. After admixture of the sample to be assayed and the antigen-antibody complex-forming reaction is completed, an enzyme-labeled antibody is added and incubated with the complex resulting from the first admixture. An excess of the labeled antibody combines with the available determinants on the antigen. Excess labeled antibody is removed by washing and enzyme activity is determined. As before, the amount of enzyme bound to the complex is an indirect measure of the amount of antigen in the assayed sample.
The term "homogeneous immunoassay" can be applied to any immunoassay system in which both the immunological reaction itself, and the detection of the extent to which the immunological reaction has occurred, are carried out in homogeneous solution, that is, without the use of a physical separation of the free and antibody-bound components. Three components are usually required. Specifically, a particular antibody, a labeled antigen and a sample that contains an unknown amount of the antigen. It is necessary that the signal arising from the label is modified, directly or indirectly, upon binding to the antibody.
Labels that produce modified signals upon binding in immune complexes include free radicals [Schneider et al., "Use of Enzyme and Spin Labelling in Homogeneous Immunochemical Detection Methods, " in Immunoassays for Drugs Subject to Abuse, Mule et al., eds., 45, CRC Press, Boca Raton, Fla., (1974)]; fluorescent dyes such as fluorescein [Ullman, E. F., Clin. Chem. (Winston-Salem), 24, 973 (1978)]; enzymes such as horseradish peroxidase (HRP) and glucose oxidase [Rubenstein et al., Biochem. Biophys. Res. Comm., 47, 846 (1972); bacteriophages [Haimovich et al., Isr. J. Med. Sci., 5, 438 (1969)]; coenzymes [Carrico et al., Anal. Biochem., 72, 271 (1976)]and phospholipid vesicles [Kinsky, S.C., Methods Enzymol., 328, 501 (1974)].
Homogeneous immunoassays are most often performed simply by mixing the sample with the reagents and measuring the signal produced by the label. Elimination of the step required in a radioimmunoassay of physically separating the free label from that which is bound to the antibody provides an important simplification and avoids a major source of imprecision in the assay. On the other hand, the main advantages of such extraction or separation steps, namely a reduction in the background signal and the elimination of interfering substances, is sacrificed. One clear advantage is that homogeneous immunoassays can be easily automated.
The specificity of the molecular binding site of an antibody is termed its idiotype. The term idiotype denotes the unique variable (V) region sequences produced by each type of antibody-forming cell. An antibody whose binding site specificity is for the binding site of another antibody is termed an anti-idiotype antibody and can be regarded as an immunologic marker for the antibody combining site. Thus, as used herein, an anti-idiotype antibody can be one form of anti-receptor antibody.
The term "cross-reactivity" refers to the ability of an antibody to bind antigens other than its idio-specific antigen. Cross-reactive anti-idiotype antibodies can be divided into two major groups. One group comprises those anti-idiotype antibodies that recognize antigenic determinants that are associated with specific amino acid sequences in the heavy and light chain variable regions. Anti-idiotype antibodies of this group often reflect the action of inherited immunoglobulin structural genes. Consequently, these antibodies do not cross-react in subjects that are not genetically similar.
The second group includes anti-idiotype antibodies that are of cross-reactive and anti-idiotype antibodies the "internal image" of the antigen. The antigenic site recognized by this group of anti-idiotype antibodies is not associated with a particular light or heavy chain amino acid sequence. Because the antibody binding site bears the internal image of the antigen; i.e. mimics the size, shape, change and/or van der Waals attraction of the antigen, this group of anti-idiotype antibody binds to many different antibodies of the same specificity. The idiotypes recognized by such antibodies can be produced by individuals with different genetic backgrounds and are controlled by genes that bear no special relationship. Several investigators have prepared internal image anti-idiotype antibodies.
For the most part, antibody response is directed against invading organisms and altered self cells or self antigens. For example, rheumatoid factor (RF) is an IgM or IgG antibody with specificity for the Fc fragment of IgG. Rheumatoid factor usually results from an abnormal condition and causes synovial inflamation and vascolitis. IgM-RF is abundant in the sera of most individuals with rheumatoid arthritis. IgM-RFs in rheumatoid sera are polyclonal and react with a number of different antigenic determinants in the Fc region of IgG. IgM-RF from unrelated individuals show cross-reactive idiotypes.
Several laboratory tests are now available to detect rheumatoid factor. The earliest test, now rarely used, was the streptococcal agglutination reaction.
The latex fixation test is now the most commonly used method for detecting rheumatoid factor. Aggregated gamma-globulin (Cohn Fraction II) is adsorbed onto latex particles, which agglutinate (clump) in the presence of rheumatoid factor. The latex fixation test is not specific for RF, but is very sensitive, resulting in a high incidence of false positive results.
The sensitized sheep red cell test (Rose-Waaler test) depends on specific antibody binding and is the most specific test in common use. Sheep red blood cells are coated with rabbit IgG antibody specific for sheep red blood cells, and the sensitized sheep cells agglutinate in the presence of rheumatoid factor.
More complicated tests include a radioimmunoassay for IgM rheumatoid factor and an immunodiffusion assay, the latter of which provides better quantification and more precise information on the immunoglobulin classes of rheumatoid factor.
Like any other antibody, anti-idiotype antibodies can be raised against IgM-RF. Anti-idiotype antibodies bearing the IgG-Fc internal image of RF react specifically with the majority of IgM-RF antibodies from rheumatoid arthritis patients. As previously stated, this is because the binding site is not associated with a particular light or heavy chain amino acid sequence, but rather because it resembles the internal image of the IgG Fc.
IgM-RFs are regularly induced in normal humans by polyclonal B cell activation. IgM-RF production eventually terminates following the withdrawal or elimination of the inducing stimulus. The factors responsible for sustained IgM-RF response are not yet fully understood. A network of idiotype and anti-idiotype antibodies has been hypothesized to modulate antibody response. It is believed that such a mechanism of idiotype and anti-idiotype immunomodulation may play some role in controlling the synthesis of RF antibodies.
Thus, it would be desirable to identify and characterize cross-reactive RF anti-idiotype antibodies. In order to do on this, it would be advantageous to have a simple procedure to purify RF anti-idiotype antibodies. Also, it would also be advantageous to have a general procedure for the purification of cross reactive anti-idiotype antibodies for other pathogenic auto-antibodies. Currently, no simple methods for purifying anti-idiotype antibodies are known.
The present invention, however, includes a method for the detection and purification of anti-receptor antibodies and (in a particular embodiment) anti-idiotype antibodies.